Treatment of Neurodegeneration and Neuroinflammation

ABSTRACT

Methods of treating a subject having a condition characterized by at least one of neurodegeneration and neuroin-flammation are provided. Methods of reducing astrogliosis in a subject having a condition characterized by increased astrogliosis are also provided. Methods of providing neuroprotection to a subject in need thereof are also provided.

This applications claims priority to U.S. Provisional Patent ApplicationNos. 61/173,797, filed Apr. 29, 2009, and 61/175,270, filed May 4, 2009.The entire disclosures of both of those applications are herebyincorporated herein by reference.

Provided are methods of treating a subject having a conditioncharacterized by at least one of neurodegeneration andneuroinflammation, by administering to the subject a therapeuticallyeffective amount of at least one compound of Formula I, or apharmaceutically acceptable salt thereof. Methods of reducingastrogliosis in a subject having a condition characterized by increasedastrogliosis, the method comprising administering to the subject atherapeutically effective amount of at least one compound of Formula Ior a pharmaceutically acceptable salt thereof are also provided. Methodsof providing neuroprotection to a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of at least one compound of Formula I or a pharmaceuticallyacceptable salt thereof are also provided. Screening methods to identifya compound as a candidate agent to treat a condition characterized by atleast one of neurodegeneration and neuroinflammation are also provided.

Astrocytes are the major cellular component of the brain. These glialcells account for about 90% of the overall brain mass and outnumberneurons five- to ten-fold in the human adult brain. In the early 1980s,two types of astrocytes were characterized: fibrous and protoplasmic.Fibrous astrocytes (type 1) have a star-like shape, are normally foundin white matter, and have long processes that run between myelinatedfibers, blood capillaries, and form vascular end-feet structures aroundthe blood-brain barrier (BBB). Conversely, protoplasmic astrocytes (type2) are ramified, have short processes, which envelop neuronal processesand inhabit the grey matter.

Activation of glial cells, microglia and astrocytes, has been implicatedas a mechanism contributing to the pathobiology of neurodegenerative andneuroinflammatory diseases, including multiple sclerosis (MS). In earlystages of disease, astrocytes can secrete cytokines and chemokines thatrecruit inflammatory cells. As the disease progresses, astrogliosis isthought to contribute to glial scarring, axonal damage, anddemyelination. Microglia have been shown to have a critical role in thedevelopment and progression of EAE pathogenesis. Pro-inflammatorycytokines produced by microglia exacerbate disease and chemokinesrecruit leukocytes to sites of inflammation. Dimethyl fumarate (DMF) isthe active component of the experimental therapeutic BG00012 currentlyin Phase III relapsing-remitting MS (RRMS) clinical trials. In the PhaseIIb RRMS study, BG00012 significantly reduced gadolinium-enhancing brainlesions. In preclinical studies, DMF has been shown to inhibit CNSinflammation in murine and rat EAE. It has now been found that DMF caninhibit astrogliosis and microglial activations associated with EAE.

Certain non-limiting aspects of the role of microglia and astrocytes inneuroinflammatory pathogenesis are shown in FIG. 2. Among the evidencefor a role of astrogliosis in neurodegeneration and neuroinflammation isevidence from the study of Multiple Sclerosis (MS), one example of adisease characterized by neurodegeneration and neuroinflammation. Inthat model activation of astrocytes and microglia occurs prior to theonset of disease symptoms and axonal damage in rodent MS models.Additionally, selective ablation of microglia reduces EAE diseaseseverity and inflammation. Clinical evidence from MS patients providesfurther evidence for a role of astrocites, because astrogliosisincreases during disease flares. Additionally, activated astrocytes havebeen reported as a prominent cell type in secondary progressive MS, andre-activation of microglia is implicated as a driver of MS diseaseflares.

Multiple sclerosis (MS) is an autoimmune disease with the autoimmuneactivity directed against central nervous system (CNS) antigens. Thedisease is characterized by inflammation in parts of the CNS, leading tothe loss of the myelin sheathing around neuronal axons (demyelination),axonal loss, and the eventual death of neurons, oligodenrocytes andglial cells. For a comprehensive review of MS and current therapies,see, e.g., McAlpine's Multiple Sclerosis, by Alastair Compston et al.,4th edition, Churchill Livingstone Elsevier, 2006.

An estimated 2,500,000 people in the world suffer from MS. It is one ofthe most common diseases of the CNS in young adults. MS is a chronic,progressing, disabling disease, which generally strikes its victims sometime after adolescence, with diagnosis generally made between 20 and 40years of age, although onset may occur earlier. The disease is notdirectly hereditary, although genetic susceptibility plays a part in itsdevelopment. MS is a complex disease with heterogeneous clinical,pathological and immunological phenotype.

There are four major clinical types of MS: 1) relapsing-remitting MS(RR-MS), characterized by clearly defined relapses with full recovery orwith sequelae and residual deficit upon recovery; periods betweendisease relapses characterized by a lack of disease progression; 2)secondary progressive MS (SP-MS), characterized by initial relapsingremitting course followed by progression with or without occasionalrelapses, minor remissions, and plateaus; 3) primary progressive MS(PP-MS), characterized by disease progression from onset with occasionalplateaus and temporary minor improvements allowed; and 4) progressiverelapsing MS (PR-MS), characterized by progressive disease onset, withclear acute relapses, with or without full recovery; periods betweenrelapses characterized by continuing progression.

Clinically, the illness most often presents as a relapsing-remittingdisease and, to a lesser extent, as steady progression of neurologicaldisability. Relapsing-remitting MS (RR-MS) presents in the form ofrecurrent attacks of focal or multifocal neurologic dysfunction. Attacksmay occur, remit, and recur, seemingly randomly over many years.Remission is often incomplete and as one attack follows another, astepwise downward progression ensues with increasing permanentneurological deficit. The usual course of RR-MS is characterized byrepeated relapses associated, for the majority of patients, with theeventual onset of disease progression. The subsequent course of thedisease is unpredictable, although most patients with arelapsing-remitting disease will eventually develop secondaryprogressive disease. In the relapsing-remitting phase, relapsesalternate with periods of clinical inactivity and may or may not bemarked by sequelae depending on the presence of neurological deficitsbetween episodes: Periods between relapses during therelapsing-remitting phase are clinically stable. On the other hand,patients with progressive MS exhibit a steady increase in deficits, asdefined above and either from onset or after a period of episodes, butthis designation does not preclude the further occurrence of newrelapses.

MS pathology is, in part, reflected by the formation of focalinflammatory demyelinating lesions in the white matter, which are thehallmarks in patients with acute and relapsing disease. In patients withprogressive disease, the brain is affected in a more global sense, withdiffuse but widespread (mainly axonal) damage in the normal appearingwhite matter and massive demyelination also in the grey matter,particularly, in the cortex.

Most current therapies for MS are aimed at the reduction of inflammationand suppression or modulation of the immune system. As of 2006, theavailable treatments for MS reduce inflammation and the number of newepisodes but not all of the treatments have an effect on diseaseprogression. A number of clinical trials have shown that the suppressionof inflammation in chronic MS rarely significantly limits theaccumulation of disability through sustained disease progression,suggesting that neuronal damage and inflammation are independentpathologies. Thus, in advanced stages of MS, neurodegeneration appearsto progress even in the absence of significant inflammation. Therefore,slowing demyelination, or promoting CNS remyelination as a repairmechanism, or otherwise preventing axonal loss and neuronal death aresome of the important goals for the treatment of MS, especially, in thecase of progressive forms of MS such as SP-MS.

Fumaric acid esters, such as dimethyl fumarate (DMF), have beenpreviously proposed for the treatment of MS (see, e.g., Schimrigk etal., Eur. J. Neurol., 2006, 13(6):604-10; Drugs R&D, 2005, 6(4):229-30;U.S. Pat. No. 6,436,992).

Provided herein is evidence that dimethyl fumarate (DMF) reducesastrocyte activation in vivo and in vitro and that DMF inhibitsinflammatory cytokines and pro-inflammatory signaling induced bylipopolysaccharide (LPS) stimulation of primary astrocytes.

Provided are methods of treating a subject having a conditioncharacterized by at least one symptom chosen from neurodegeneration andneuroinflammation. In some embodiments the method includes administeringto the subject a therapeutically effective amount of at least onecompound of Formula I:

wherein R¹ and R² are independently selected from OH, O⁻, and(C₁₋₆)alkoxy, provided that at least one of R¹ and R² is (C₁₋₆)alkoxy,or a pharmaceutically acceptable salt thereof. In some embodiments, theadministration to the subject of the therapeutically effective amount ofthe at least one compound of Formula I, or a pharmaceutically acceptablesalt thereof, results in the supression of expression in the subject ofat least one gene selected from Ccl20, Ccl3, Ccl4, Cxcl1, Cxcl10, Cxcl2,Cxcl3, Cxcl6, IL1a, Mb, Tnf, Ifit3, Nfkbia, Nfkbiz, Tnfaip2, andZc3h12a. In some embodiments, the condition characterized by at leastone symptom chosen from neurodegeneration and neuroinflammation isfurther characterized by increased expression of at least one geneselected from Ccl20, Ccl3, Ccl4, Cxcl1, Cxcl10, Cxcl2, Cxcl3, Cxcl6,IL1a, Mb, Tnf, Ifit3, Nfkbia, Nfkbiz, Tnfaip2, and Zc3h12a. In someembodiments, the administration to the subject of the therapeuticallyeffective amount of the at least one compound of Formula I results inupregulation of expression in the subject of at least one gene selectedfrom Gsta2, Gsta3, Gcic, Ggt1, Txnrd1, Srxn1, Sqstm1, and Nqo1. In someembodiments increased expression of at least one gene selected fromGsta2, Gsta3, Gcic, Ggt1, Txnrd1, Srxn1, Sqstm1, and Nqo1 is achieved inthe absence of supression of expression in the subject of at least onegene selected from Ccl20, Ccl3, Ccl4, Cxcl1, Cxcl10, Cxcl2, Cxcl3,Cxcl6, IL1a, II1b, Tnf, Ifit3, Nfkbia, Nfkbiz, Tnfaip2, and Zc3h12a. Insome embodiments, the at least one compound is formulated as apharmaceutical composition comprising the at least one compound and atleast one pharmaceutically acceptable vehicle chosen from carriers,adjuvants, and excipients. In some embodiments, the at least onecompound is chosen from dimethyl fumarate and monomethyl fumarate. Insome embodiments, the only active agent administered to the subject isdimethyl fumarate (DMF). In some embodiments, the only active agentadministered to the subject is monomethyl fumarate (MMF). In someembodiments, the only active agents administered to the subject are DMFand MMF. In some embodiments, the at least one compound is administeredin an amount and for a period of time sufficient to reduce at least oneof neurodegeneration and neuroinflammation in the subject. In someembodiments, the condition characterized by at least one symptom chosenfrom neurodegeneration and neuroinflammation is selected from AdrenalLeukodystrophy (ALD), Alcoholism, Alexander's disease, Alper's disease,Alzheimer's disease, Amyotrophic lateral sclerosis (Lou Gehrig'sDisease), Ataxia telangiectasia, Batten disease (also known asSpielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiformencephalopathy (BSE), Canavan disease, Cerebral palsy, Cockaynesyndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, FamilialFatal Insomnia, Frontotemporal lobar degeneration, Huntington's disease,HIV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy bodydementia, Neuroborreliosis, Machado-Joseph disease (Spinocerebellarataxia type 3), Multiple System Atrophy, Multiple sclerosis, Narcolepsy,Niemann Pick disease, Parkinson's disease, Pelizaeus-Merzbacher Disease,Pick's disease, Primary lateral sclerosis, Prion diseases, ProgressiveSupranuclear Palsy, Refsum's disease, Sandhoff disease, Schilder'sdisease, Subacute combined degeneration of spinal cord secondary toPernicious Anaemia, Spielmeyer-Vogt-Sjogren-Batten disease (also knownas Batten disease), Spinocerebellar ataxia, Spinal muscular atrophy,Steele-Richardson-Olszewski disease, Tabes dorsalis, Toxicencephalopathy, LHON (Leber's Hereditary optic neuropathy), MELAS(Mitochondrial Encephalomyopathy; Lactic Acidosis; Stroke), MERRF(Myoclonic Epilepsy; Ragged Red Fibers), PEO (Progressive ExternalOpthalmoplegia), Leigh's Syndrome, MNGIE (Myopathy and externalophthalmoplegia; Neuropathy; Gastro-Intestinal; Encephalopathy),Kearns-Sayre Syndrome (KSS), NARP, Hereditary Spastic Paraparesis,Mitochondrial myopathy, and Friedreich Ataxia. In some embodiments, thecondition characterized by at least one of neurodegeneration andneuroinflammation is Multiple Sclerosis (MS). In some embodiments, thesubject does not have MS. In some embodiments the subject has relapsingremitting multiple sclerosis and treatment reduces the frequency ofclinical relapses and delays the accumulation of physical disability. Insome embodiments the subject has relapsing remitting multiple sclerosisand treatment reduces the frequency of clinical exacerbations.

Also provided are methods of reducing astrogliosis in a subject having acondition characterized by increased astrogliosis. In some embodimentsthe methods include administering to the subject a therapeuticallyeffective amount of at least one compound of Formula I:

wherein R¹ and R² are independently selected from OH, O⁻, and(C₁₋₆)alkoxy, or a pharmaceutically acceptable salt thereof. In someembodiments, the administration to the subject of the therapeuticallyeffective amount of the at least one compound of Formula I, or apharmaceutically acceptable salt thereof, results in the supression ofexpression in the subject of at least one gene selected from Ccl20,Ccl3, Ccl4, Cxcl1, Cxcl10, Cxcl2, Cxcl3, Cxcl6, IL1a, Mb, Tnf, Ifit3,Nfkbia, Nfkbiz, Tnfaip2, and Zc3h12a. In some embodiments, theadministration to the subject of the therapeutically effective amount ofthe at least one compound of Formula I results in upregulation ofexpression in the subject of at least one gene selected from Gsta2,Gsta3, Gcic, Ggt1, Txnrd1, Srxn1, Sqstm1, and Nqo1. In some embodimentsincreased expression of at least one gene selected from Gsta2, Gsta3,Gclc, Ggt1, Txnrd1, Srxn1, Sqstm1, and Nqo1 is achieved in the absenceof supression of expression in the subject of at least one gene selectedfrom Ccl20, Ccl3, Ccl4, Cxcl1, Cxcl10, Cxc12, Cxcl3, Cxcl6, IL1a, Mb,Tnf, Ifit3, Nfkbia, Nfkbiz, Tnfaip2, and Zc3h12a. In some embodiments,the at least one compound is formulated as a pharmaceutical compositioncomprising the at least one compound and at least one pharmaceuticallyacceptable vehicle chosen from carriers, adjuvants, and excipients. Insome embodiments, the at least one compound is formulated as apharmaceutical composition comprising the at least one compound and atleast one pharmaceutically acceptable vehicle chosen from carriers,adjuvants, and excipients. In some embodiments, the at least onecompound is chosen from dimethyl fumarate and monomethyl fumarate. Insome embodiments, the only active agent administered to the subject isdimethyl fumarate (DMF). In some embodiments, the only active agentadministered to the subject is monomethyl fumarate (MMF). In someembodiments, the only active agents administered to the subject are DMFand MMF. In some embodiments, the at least one compound is administeredin an amount and for a period of time sufficient to reduce at least oneof neurodegeneration and neuroinflammation in the subject. In someembodiments, the condition characterized by increased astrogliosis isselected from Adrenal Leukodystrophy (ALD), Alcoholism, Alexander'sdisease, Alper's disease, Alzheimer's disease, Amyotrophic lateralsclerosis (Lou Gehrig's Disease), Ataxia telangiectasia, Batten disease(also known as Spielmeyer-Vogt-Sjogren-Batten disease), Bovinespongiform encephalopathy (BSE), Canavan disease, Cerebral palsy,Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease,Familial Fatal Insomnia, Frontotemporal lobar degeneration, Huntington'sdisease, HIV-associated dementia, Kennedy's disease, Krabbe's disease,Lewy body dementia, Neuroborreliosis, Machado-Joseph disease(Spinocerebellar ataxia type 3), Multiple System Atrophy, Multiplesclerosis, Narcolepsy, Niemann Pick disease, Parkinson's disease,Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral sclerosis,Prion diseases, Progressive Supranuclear Palsy, Refsum's disease,Sandhoff disease, Schilder's disease, Subacute combined degeneration ofspinal cord secondary to Pernicious Anaemia,Spielmeyer-Vogt-Sjogren-Batten disease (also known as Batten disease),Spinocerebellar ataxia, Spinal muscular atrophy,Steele-Richardson-Olszewski disease, Tabes dorsalis, Toxicencephalopathy, LHON (Leber's Hereditary optic neuropathy), MELAS(Mitochondrial Encephalomyopathy; Lactic Acidosis; Stroke), MERRF(Myoclonic Epilepsy; Ragged Red Fibers), PEO (Progressive ExternalOpthalmoplegia), Leigh's Syndrome, MNGIE (Myopathy and externalophthalmoplegia; Neuropathy; Gastro-Intestinal; Encephalopathy),Kearns-Sayre Syndrome (KSS), NARP, Hereditary Spastic Paraparesis,Mitochondrial myopathy, and Friedreich Ataxia. In some embodiments, thecondition characterized by at least one of neurodegeneration andneuroinflammation is Multiple Sclerosis (MS). In some embodiments, thesubject does not have MS. In some embodiments the subject has relapsingremitting multiple sclerosis and treatment reduces the frequency ofclinical relapses and delays the accumulation of physical disability. Insome embodiments the subject has relapsing remitting multiple sclerosisand treatment reduces the frequency of clinical exacerbations

Also provided are methods of identifying a compound as a candidateneuroprotection agent. In some embodiments the methods include a)inducing at least one of neurodegeneration and neuroinflammation in atarget cell, tissue, or mammal, b) measuring expression of at least onemarker of at least one of neurodegeneration and neuroinflammation in thetarget cell, tissue, or mammal in the presence of the compound, and c)measuring expression of at least one marker of at least one ofneurodegeneration and neuroinflammation in the target cell, tissue, ormammal in the absence of the compound, wherein, if the expression of atleast one marker of at least one of neurodegeneration andneuroinflammation is reduced in the presence of the compound relative toits expression in the absence of the compound, the compound isidentified as a candidate neuroprotection agent. In some embodiments themethods further comprise d) measuring astrogliosis in the presence ofthe at least one compound, and e) measuring astrogliosis in the absenceof the at least once compound, wherein, astrogliosis is reduced in thepresence of the compound relative to the level of astrogliosis in theabsence of the compound. In some embodiments the at least one marker isthe expression level of at least one gene selected from Ccl20, Ccl3,Ccl4, Cxcl1, Cxcl10, Cxcl2, Cxcl3, Cxcl6, IL1a, II1b, Tnf, Ifit3,Nfkbia, Nfkbiz, Tnfaip2, and Zc3h12a.

Also provided are methods of providing neuroprotection to a subject inneed thereof. In some embodiments the methods include administering tothe subject a therapeutically effective amount of at least one compoundof Formula I:

wherein R¹ and R² are independently selected from OH, O⁻, and(C₁₋₆)alkoxy, provided that at least one of R¹ and R² is (C₁₋₆)alkoxy,or a pharmaceutically acceptable salt thereof, wherein theadministration to the subject of the therapeutically effective amount ofthe at least one compound of Formula I, or a pharmaceutically acceptablesalt thereof, results in the supression of expression in the subject ofat least one gene selected from Ccl20, Ccl3, Ccl4, Cxcl1, Cxcl10, Cxcl2,Cxcl3, Cxcl6, IL1a, Mb, Tnf, Ifit3, Nfkbia, Nfkbiz, Tnfaip2, andZc3h12a. In some embodiments, the the condition characterized by atleast one symptom chosen from neurodegeneration and neuroinflammation isfurther characterized by increased expression of at least one geneselected from Ccl20, Ccl3, Ccl4, Cxcl1, Cxcl10, Cxcl2, Cxcl3, Cxcl6,IL1a, IIIb, Tnf, Ifit3, Nfkbia, Nfkbiz, Tnfaip2, and Zc3h12a. In someembodiments, the administration to the subject of the therapeuticallyeffective amount of the at least one compound of Formula I results inupregulation of expression in the subject of at least one gene selectedfrom Gsta2, Gsta3, Gcic, Ggt1, Txnrd1, Srxn1, Sqstm1, and Nqo1. In someembodiments increased expression of at least one gene selected fromGsta2, Gsta3, Gcic, Ggt1, Txnrd1, Srxn1, Sqstm1, and Nqo1 is achieved inthe absence of supression of expression in the subject of at least onegene selected from Ccl20, Ccl3, Ccl4, Cxcl 1, Cxcl10, Cxcl2, Cxcl3,Cxcl6, IL1a, II1b, Tnf, Ifit3, Nfkbia, Nfkbiz, Tnfaip2, and Zc3h12a. Insome embodiments, the at least one compound is formulated as apharmaceutical composition comprising the at least one compound and atleast one pharmaceutically acceptable vehicle chosen from carriers,adjuvants, and excipients. In some embodiments, the at least onecompound is chosen from dimethyl fumarate and monomethyl fumarate. Insome embodiments, the only active agent administered to the subject isdimethyl fumarate (DMF). In some embodiments, the only active agentadministered to the subject is monomethyl fumarate (MMF). In someembodiments, the only active agents administered to the subject are DMFand MMF. In some embodiments, the at least one compound is administeredin an amount and for a period of time sufficient to reduce at least oneof neurodegeneration and neuroinflammation in the subject. In someembodiments, the condition characterized by at least one symptom chosenfrom neurodegeneration and neuroinflammation is selected from AdrenalLeukodystrophy (ALD), Alcoholism, Alexander's disease, Alper's disease,Alzheimer's disease, Amyotrophic lateral sclerosis (Lou Gehrig'sDisease), Ataxia telangiectasia, Batten disease (also known asSpielmeyer-Vogt-Sjögren-Batten disease), Bovine spongiformencephalopathy (BSE), Canavan disease, Cerebral palsy, Cockaynesyndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, FamilialFatal Insomnia, Frontotemporal lobar degeneration, Huntington's disease,HIV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy bodydementia, Neuroborreliosis, Machado-Joseph disease (Spinocerebellarataxia type 3), Multiple System Atrophy, Multiple sclerosis, Narcolepsy,Niemann Pick disease, Parkinson's disease, Pelizaeus-Merzbacher Disease,Pick's disease, Primary lateral sclerosis, Prion diseases, ProgressiveSupranuclear Palsy, Refsum's disease, Sandhoff disease, Schilder'sdisease, Subacute combined degeneration of spinal cord secondary toPernicious Anaemia, Spielmeyer-Vogt-Sj ogren-Batten disease (also knownas Batten disease), Spinocerebellar ataxia, Spinal muscular atrophy,Steele-Richardson-Olszewski disease, Tabes dorsalis, Toxicencephalopathy, LHON (Leber's Hereditary optic neuropathy), MELAS(Mitochondrial Encephalomyopathy; Lactic Acidosis; Stroke), MERRF(Myoclonic Epilepsy; Ragged Red Fibers), PEO (Progressive ExternalOpthalmoplegia), Leigh's Syndrome, MNGIE (Myopathy and externalophthalmoplegia; Neuropathy; Gastro-Intestinal; Encephalopathy),Keams-Sayre Syndrome (KSS), NARP, Hereditary Spastic Paraparesis,Mitochondrial myopathy, and Friedreich Ataxia. In some embodiments, thecondition characterized by at least one of neurodegeneration andneuroinflammation is Multiple Sclerosis (MS). In some embodiments, thesubject does not have MS. In some embodiments the subject has relapsingremitting multiple sclerosis and treatment reduces the frequency ofclinical relapses and delays the accumulation of physical disability. Insome embodiments the subject has relapsing remitting multiple sclerosisand treatment reduces the frequency of clinical exacerbations.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A: DMF dose response in a rat EAE model.

FIG. 1B: Glial cell inhibition by BG00012.

FIG. 2: Various molecular mediators of the roles of astrocytes andmicroglia in neuroinflammatory pathogenesis.

FIG. 3A: Astrocyte staining of rat spinal cords with and without DMFtreatment.

FIG. 3B: Morphometric quantitation using Aperio color deconvolution.

FIG. 3C: Ventral grey matter and white matter regions.

FIG. 3D: Morphometric quantitation of positive GFAP staining in ventralgrey and white matter.

FIG. 4A: BG200012 inhibits expression of GFAP.

FIG. 4B: BG200012 inhibits LPS induced TNF expression.

FIG. 4C: In vitro PD response to BG00012 in astrocytes.

FIG. 4D: Astrocyte viability following BG00012 treatment.

FIG. 5: BG00012 inhibits inflammatory cytokines and pro-inflammatorysignaling induced by LPS stimulation of primary astrocytes.

FIG. 6A: Glutathione levels in cultured astrocytes.

FIG. 6B: Metabolic activity of cultured astrocytes.

FIG. 6C: Cell viability of cultured astrocytes.

FIG. 7A: Raw fluorescence traces from cells treated with MMF.

FIG. 7B: Ca⁺⁺ mobilization in cells treated with MMF.

FIG. 7C: Non-linear regression analysis of data.

FIG. 8A: Cell viability of cultures astrocytes.

FIG. 8B: Metabolic activity of cultured astrocytes.

FIG. 8C: ATP levels in cultured astrocytes.

FIG. 9: DMF and MMF increase cellular levels of Nrf2 in primary rat andhuman asrtrocytes.

FIG. 10: Canonical signaling pathways stimulated by DMF in primary ratastrocytes.

FIG. 11: Major cellular functions affected by DMF in primary rasastrocytes.

FIG. 12: DMF treatment diminishes myelin loss during EAE (rat spinalcords).

A condition characterized by at least one of neurodegeneration andneuroinflammation is a condition in which either or both of thoseprocesses leads to a failure of the subjects nervous system to functionnormally. The loss of normal function may be located in either or bothof the central nervous system (e.g., the brain, spinal cord) and theperipheral nervous system. Examples of such conditions include, but arenot limed to, Adrenal Leukodystrophy (ALD), Alcoholism, Alexander'sdisease, Alper's disease, Alzheimer's disease, Amyotrophic lateralsclerosis (Lou Gehrig's Disease), Ataxia telangiectasia, Batten disease(also known as Spielmeyer-Vogt-Sjögren-Batten disease), Bovinespongiform encephalopathy (BSE), Canavan disease, Cerebral palsy,Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease,Familial Fatal Insomnia, Frontotemporal lobar degeneration, Huntington'sdisease, HIV-associated dementia, Kennedy's disease, Krabbe's disease,Lewy body dementia, Neuroborreliosis, Machado-Joseph disease(Spinocerebellar ataxia type 3), Multiple System Atrophy, Multiplesclerosis, Narcolepsy, Niemann Pick disease, Parkinson's disease,Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral sclerosis,Prion diseases, Progressive Supranuclear Palsy, Refsum's disease,Sandhoff disease, Schilder's disease, Subacute combined degeneration ofspinal cord secondary to Pernicious Anaemia,Spielmeyer-Vogt-Sjogren-Batten disease (also known as Batten disease),Spinocerebellar ataxia, Spinal muscular atrophy,Steele-Richardson-Olszewski disease, Tabes dorsalis, Toxicencephalopathy, LHON (Leber's Hereditary optic neuropathy), MELAS(Mitochondrial Encephalomyopathy; Lactic Acidosis; Stroke), MERRF(Myoclonic Epilepsy; Ragged Red Fibers), PEO (Progressive ExternalOpthalmoplegia), Leigh's Syndrome, MNGIE (Myopathy and externalophthalmoplegia; Neuropathy; Gastro-Intestinal; Encephalopathy),Kearns-Sayre Syndrome (KSS), NARP, Hereditary Spastic Paraparesis,Mitochondrial myopathy, and Friedreich Ataxia.

In some embodiments, administration of at least one compound orpharmaceutically acceptable salt thereof, as described herein, to apatient gives rise to “neuroprotection,” or said another way, the effectof administering the compound to the patient is neuroprotection.Neuroprotection comprises at least one of maintenance, salvage,recovery, and regeneration of the nervous system, its cells, structure,and function following injury or damage. In some embodimentsneuroprotection comprises at least one of primary neuroprotection andsecondary neuroprotection. “Primary neuroprotection” is protectioncomprising direct modulation of the structure and/or function of neuralcells residing within the CNS (at least one cell type selected fromneurons, oligodendrocytes, astrocytes, and microglia). “Secondaryneuroprotection” is protection comprising modulation of the structure orfunction of at least one cell type that typically resides outside theCNS (e.g. immune cells). In secondary neuroprotection the at least onecompound or pharmaceutically acceptable salt thereof acts directly orindirectly on the at least one cell type that typically resides outsidethe CNS to modulate the structure and/or function of that at least onecell type. That at least one cell type then modulates, directly orotherwise, the structure and/or function of neural cells residing withinthe CNS (at least one cell type selected from neurons, oligodendrocytes,astrocytes, and microglia). In some embodiments, neuroprotectioncomprises a lessening of the severity or rate of neurodegeneration orneuroinflammation in a subject. “Maintenance” of the nervous system, itscells, structure, and function comprises embodiments in which the atleast one compound or pharmaceutically acceptable salt thereof isadministered to a subject prior to development of at least one sign orsymptom of a disease or condition disclosed herein and reduces theeventual severity of the disease or condition and/or reduces the rate ofonset of the disease and/or condition.

In some embodiments the condition to be treated is characterized byincreased expression of pro-inflammatory genes, such as in neural cellsof the subject. In the case of a subject experiencing astrogliosis, forexample, expression of at least one pro-inflammatory gene selected fromCcl20, Ccl3, Ccl4, Cxcl1, Cxcl10, Cxcl2, Cxcl3, Cxcl6, IL1a, IIIb, Tnf,Ifit3, Nfkbia, Nfkbiz, Tnfaip2, and Zc3h12a is increased in the subject.In some embodiments, administration of at least one compound orpharmaceutically acceptable salt thereof, as described herein, to thesubject, results in suppression of expression of at least one geneselected from Ccl20, Ccl3, Ccl4, Cxcl1, Cxcl10, Cxcl2, Cxcl3, Cxcl6,IL1a, II1b, Tnf, Ifit3, Nfkbia, Nfkbiz, Tnfaip2, and Zc3h12a.

Certain examples of neuroprotective genes are also disclosed herein,namely Gsta2, Gsta3, Gcic, Ggt1, Txnrd1, Srxn1, Sqstm1, and Nqo1. Insome embodiments, administration of at least one compound orpharmaceutically acceptable salt thereof, as described herein, to thesubject, results in upregulation of at least one gene selected fromGsta2, Gsta3, Gcic, Ggt1, Txnrd1, Srxn1, Sqstm1, and Nqo1.

The term “therapeutically effective amount” refers to that amount of acompound or pharmaceutically acceptable salt thereof which results inprevention or delay of onset or amelioration of at least one symptom ofa condition characterized by neurodegeneration or neuroinflammation in asubject, or an attainment of a desired biological outcome, such asreduced astrogliosis.

In some embodiments the expression level of at least one gene selectedfrom Ccl20, Ccl3, Ccl4, Cxcl1, Cxcl10, Cxcl2, Cxcl3, Cxcl6, IL1a, II1b,Tnf, Ifit3, Nfkbia, Nfkbiz, Tnfaip2, Zc3h12a, Gsta2, Gsta3, Gcic, Ggt1,Txnrd1, Srxn1, Sqstm1, and Nqo1 is measured in a subject. In someembodiments, expression of the gene is measured by determining theexpression level of an mRNA for that gene. In some embodiments,expression of the gene is measured by determining the expression levelof a protein product encoded by the gene. In some embodiments theprotein product is measured in cerebrospinal fluid of the subject. Insome embodiments expression level is measured at at least one time pointselected from prior to initiation of treatment, during treatment, andafter treatment.

The term “treating” refers to administering a therapy in an amount,manner, and/or mode effective to prevent or delay onset of oramelioration of at least one symptom of a condition characterized byneurodegeneration or neuroinflammation in a subject, to either astatistically significant degree or to a degree detectable to oneskilled in the art. An effective amount, manner, or mode can varydepending on the subject and may be tailored to the subject. Forneurological disorders referred herein, the treatment offered by themethod of this invention aims at improving the conditions (or lesseningthe detrimental effects) of the disorders and not necessarily atcompletely eliminating or curing the disorders.

Unless otherwise specified, the term “MMF” refers to monomethyl fumaratein the form of acid (methyl hydrogen fumarate, also known as “MHF”) aswell as to its corresponding salts.

In some embodiments, the methods of the invention comprise administeringto a subject having the condition a therapeutically effective amount ofat least one compound of Formula I:

wherein R¹ and R² are independently selected from OH, O⁻, (C₁₋₆)alkoxy,or a pharmaceutically acceptable salt thereof. (C₁₋₆)alkoxy can bechosen from, for example, (C₁₋₅)alkoxy, (C₁₋₄)alkoxy, (C₁₋₃)alkoxy,ethoxy, methoxy, (C₂₋₃)alkoxy, (C₂₋₄)alkoxy, (C₂₋₅)alkoxy, and(C₁₋₆)alkoxy. In some embodiments of the compounds of Formula I, thepharmaceutically acceptable salt is a salt of a metal (M) cation,wherein M can be an alkali, alkaline earth, or transition metal such asLi, Na, K, Ca, Zn, Sr, Mg, Fe, or Mn. In nonlimiting illustrativeembodiments, the compound of Formula I is dimethyl fumarate (R¹ is CH₃and R² is CH₃) or monomethyl fumarate (R¹ is CH₃ and R² is O⁻ or OH,e.g., a pharmaceutically acceptable salt of monomethyl fumarate, e.g.,specifically, Ca-MMF).

In certain embodiments the methods of the invention provide a subjectwith a reduction in neurodegeneration and/or neuroinflammation. Theseneuroprotective effects do not necessarily eliminate all of the damagesor degeneration, but rather, delay or even halt the progress of thedegeneration or a prevention of the initiation of the degenerationprocess or an improvement to the pathology of the disorder. The methodsof the invention may offer neuroprotection to any part of the nervoussystem, such as, the central nervous system, e.g., hippocampus,cerebellum, spinal cord, cortex (e.g., motor or somatosensory cortex),striatum, basal forebrain (cholenergic neurons), ventral mesencephalon(cells of the substantia nigra), and the locus ceruleus (neuroadrenalinecells of the central nervous system).

In some embodiments, the at least one compound or pharmaceuticallyacceptable salt thereof is administered in an amount and for a period oftime sufficient to reduce at least one of neurodegeneration andneuroinflammation in the subject. In some embodiments, the at least onecompound is administered in an amount and for a period of timesufficient to reduce astrogliosis in the subject. In some embodiments,the at least one compound or pharmaceutically acceptable salt thereof isadministered in an amount and for a period of time sufficient to provideneuroprotection to the subject.

Methods of the invention may include treating the subject with atherapeutically effective amount of at least one compound chosen fromDMF and MMF. For DMF or MMF, the therapeutically effective amount canrange from about 1 mg/kg to about 50 mg/kg (e.g., from about 2.5 mg/kgto about 20 mg/kg or from about 2.5 mg/kg to about 15 mg/kg). Effectivedoses will also vary, as recognized by those skilled in the art,dependent on route of administration, excipient usage, and thepossibility of co-usage with other therapeutic treatments including useof other therapeutic agents. For example, an effective dose of DMF orMMF to be administered to a subject, for example orally, can be fromabout 0.1 g to about 1 g per day, for example, from about 200 mg toabout 800 mg per day (e.g., from about 240 mg to about 720 mg per day;or from about 480 mg to about 720 mg per day; or about 720 mg per day).For example, 720 mg per day may be administered in separateadministrations of 2, 3, 4, or 6 equal doses.

In some embodiments of the methods 120 mg of dimethyl fumarate ispresent in the pharmaceutical preparation. In some embodiments of themethods the pharmaceutical preparation is administered to the patientthree times per day (TID). In some embodiments of the methods thepharmaceutical preparation is administered to the patient two times perday (BID).

In some embodiments of the methods 240 mg of dimethyl fumarate ispresent in the pharmaceutical preparation. In some embodiments of themethods the pharmaceutical preparation is administered to the patientthree times per day (TID). In some embodiments of the methods thepharmaceutical preparation is administered to the patient two times perday (BID).

In some embodiments of the methods the pharmaceutical preparation isadministered at least one hour before or after food is consumed by thepatient.

In some embodiments of the methods administering the pharmaceuticalpreparation further comprises administering to the patient a first doseof the pharmaceutical preparation for a first dosing period; andadministering to the patient a second dose of the pharmaceuticalpreparation for a second dosing period. In some embodiments of themethods the first dosing period is at least one week. In someembodiments of the methods the first dose of the pharmaceuticalpreparation comprises 120 mg of dimethyl fumarate and the pharmaceuticalpreparation is administered to the patient three times per day (TID) forthe first dosing period. In some embodiments of the methods the seconddose of the first pharmaceutical preparation comprises 240 mg ofdimethyl fumarate and the first pharmaceutical preparation isadministered to the patient three times per day (TID) for the seconddosing period. In some embodiments of the methods the second dose of thefirst pharmaceutical preparation comprises 240 mg of dimethyl fumarateand the first pharmaceutical preparation is administered to the patienttwo times per day (BID) for the second dosing period. In someembodiments of the methods, if the patient experiences flushing or agastrointestinal disturbance during the second dosing period then thepatient is administered a dose of the first pharmaceutical preparationcomprising 120 mg of dimethyl fumarate three times per day (TID) for aperiod of from 1 week to 1 month

The therapeutic compound (e.g., DMF or MMF) can be administered by anymethod that permits the delivery of the compound for treatment ofneurological disorders. For instance, the therapeutic compound can beadministered via pills, tablets, microtablets, pellets, micropellets,capsules (e.g., containing microtablets), suppositories, liquidformulations for oral administration, and in the form of dietarysupplements. The pharmaceutically acceptable compositions can includewell-known pharmaceutically acceptable excipients, e.g., if thecomposition is an aqueous solution containing the active agent, it canbe an isotonic saline, 5% glucose, or others. Solubilizing agents suchas cyclodextrins, or other solubilizing agents well known to thosefamiliar with the art, can be utilized as pharmaceutical excipients fordelivery of the therapeutic compound. See, e.g., U.S. Pat. Nos.6,509,376 and 6,436,992 for some formulations containing DMF and/or MMF.As to route of administration, the compositions can be administeredorally, intranasally, transdermally, subcutaneously, intradermally,vaginally, intraaurally, intraocularly, intramuscularly, buccally,rectally, transmucosally, or via inhalation, or intravenousadministration. Preferably, DMF or MMF is administered orally.

In some embodiments, a method according to the invention comprisesadministering orally a capsule containing a pharmaceutical preparationconsisting essentially of 60-240 mg (e.g., 120 mg) of dimethyl fumaratein the form of enteric-coated microtablets. In some embodiments, themean diameter of such microtablets is 1-5 mm, e.g., 1-3 mm or 2 mm.

The therapeutic compound can be administered in the form of a sustainedor controlled release pharmaceutical formulation. Such formulation canbe prepared by various technologies by a skilled person in the art. Forexample, the formulation can contain the therapeutic compound, arate-controlling polymer (i.e., a material controlling the rate at whichthe therapeutic compound is released from the dosage form) andoptionally other excipients. Some examples of rate-controlling polymersare hydroxy alkyl cellulose, hydroxypropyl alkyl cellulose (e.g.,hydroxypropyl methyl cellulose, hydroxypropyl ethyl cellulose,hydroxypropyl isopropyl cellulose, hydroxypropyl butyl cellulose andhydroxypropyl hexyl cellulose), poly(ethylene)oxide, alkyl cellulose(e.g., ethyl cellulose and methyl cellulose), carboxymethyl cellulose,hydrophilic cellulose derivatives, and polyethylene glycol, andcompositions as described in WO 2006/037342, WO 2007/042034, WO2007/042035, WO 2007/006308, WO 2007/006307, and WO 2006/050730.

In some embodiments in which dimethyl fumarate is administered to a thepatient the DMF is formulated in capsules containing enteric coatedmicrotablets. This formulation is referred to herein as “BG-12” or“BG00012”. The coating of the tablets is composed of different layers.The first layer is a methacrylic acid - methyl methacrylatecopolymer/isopropyl alcohol solution which isolates the tablet coresfrom potential hydrolysis from the next applied water suspensions.Enteric coating of the tablet is then conferred by an aqueousmethacrylic acid-ethyl acrylate copolymer suspension. The completecomponents and quantitative composition of the capsules are given inTable 1.

TABLE 1 Ingredients Amount/capsule Function Core Microtablets Activeingredients: Dimethyl Fumarate* 120.00 mg active ingredient Excipients:Croscarmellose sodium  15.00 mg disintegrant Microcrystalline Cellulose131.60 mg filler Magnesium stearate  5.00 mg lubricant Talcum  19.80 mgglidant Silica colloidal anhydrous  2.60 mg glidant Mass coremicrotablets 294.00 mg Coating Microtablets Excipients: TriethylCitrate**  7.60 mg plasticizer Methacrylic Acid-Methyl  5.50 mg filmcoating agent Methacrylate Copolymer (1:1) as Methacrylic Acid-Methyl (44.00 mg) Methacrylate Copolymer (1:1) solution 12.5%** Simeticone(corresponding to  0.17 mg anti-foam agent Simeticone Ph Eur) asSimeticone Emulsion USP**  (0.53 mg) Talcum micronised**  13.74 mglubricant Methacrylic acid-Ethyl Acrylate  33.00 mg film coating agentCopolymer (1:1) as Methacrylic acid-Ethyl (110.00 mg) Acrylate Copolymer(1:1) dispersion 30% ** Mass enteric coated microtablets 354.01 mg Massof gelatin capsule  96.00 mg Mass of filled capsule 450.01 mg

The manufacturing process and process controls include the following:

A) Active and non-active ingredients are weighed and each startingmaterial is identified with an appropriate labelling (denomination,batch number, quantity).

B) Blending: A powder mixture containing the active ingredient dimethylfumarate and all excipients of the core microtablets is prepared.

C) Tabletting: A rotative press is equipped with multiple-punches tools,a deduster and the powder mixture is tabletted according to the givenspecifications.

D) Film Coating: In accordance with commonly used film coating methodsthe microtablet cores are isolated by spraying an isolation solutionusing a film coating equipment. The isolated cores are sprayed with anenteric coating suspension in the film coating pan. Thegastro-resistance of microtablets and the active ingredient content arecontrolled.

E) Capsule Filling: Based on microtablets active ingredient the capsulesare filled with an amount corresponding to 120 mg of active ingredientper capsule. The capsule filling weight and capsule length arecontrolled.

F) Packaging: The capsules are packaged on a blistering machine inthermoformed PVC/PE/PVdC—Aluminium blisters.

Additional methods of synthesizing and formulating DMF and MMF areprovided, for example, in the Examples at columns 5-7 of U.S. Patent No.7,320,999, and in WO 2006/037342, WO 2007/042034, WO 2007/042035, WO2007/006308, WO 2007/006307, and WO 2006/050730.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

Methods

Rat EAE:

Female Brown Norway rats (Charles River Laboratories) were immunizedintradermally at the base of the tail with MOG 1-125 100 ug/rat in IFAat day 0. Day 3 DMF (BG00012) qd 5, 25, 50, 100, 200 mg/kg deliveredorally as a suspension in 0.8% HPMC. N=6 per group. Scoring 0=nodisease, 1=tail paralysis, 2=hind limb weakness, 3=hind limb paralysis,4=hind limb paralysis and forelimb weakness, 5=moribund or dead.Experimental in vivo procedures were performed in accordance withInstitutional Animal Committee guidelines

Primary Astrocyte Cultures:

Rat astrocytes from cortex, hippocampus and striatum (Lonza clontech)were cultured as described by manufacturers protocol. Limiting dilutionsof DMF in DMSO were added to cultures for 6 or 24 hours and stimulatedwith E. Coli LPS (Sigma). RNA was prepared using QIAgen Rneasy method.

Histology and Morphometry:

Lumbar spinal cord sections were prepared as FFPE sections forimmunohistochemistry and processed on DAKO autostainer using GFAPantibody (DAKO), IBA-1 (Wako), CD3 (DAKO). Aperio Spectrum ColorDeconvolution software was used for morphometric analysis.

Expression Analysis:

Total RNA was made from snap frozen lumbar spinal cord sections usingQiagen RNeasy methods. Applied Biosciences TaqMan probes were used toamplify specific transcripts and normalized using the GAPDH housekeepingprobe.

EXAMPLE 1

BG00012, an orally available formulation of dimethyl fumarate (DMF), isin Phase III testing for relapsing-remitting multiple sclerosis (RRMS).In Phase IIb testing, BG-12 significantly reduced gadalinium enhancingbrain lesions and reduced Tl hypointense black holes. The activecomponent of BG00012, dimethyl fumarate (DMF), was tested in rat EAEmodels. As shown in FIG. 1A, treatment of rats with experimentallyinduced EAE with various doses of DMF reduced EAE symptoms in adose-dependent manner. Treatment with 200 mg/kg DMF completely abrogateddisease. (FIG. 1A).

FIG. 1B shows cross sections of spinal cords of rats treated withvehicle or BG00012. The treatment and staining of the panels a-f is asfollows:

a. Luxol Fast Blue/vehicle

b . Luxol Fast Blue/BG00012

c. GFAP/vehicle

d. GFAP/BG00012

e. IBM/vehicle

f. IBM/BG0012

As shown in FIG. 1B, activated astrocytes and microglia are markedlyreduced in spinal cords treated with BG00012 and myelin is preserved.

EXAMPLE 2

This example shows that BG00012 reduces astrocyte activation in vivo.Specifically, spinal cords from DMF treated rats have fewer activatedastrocytes in they grey matter than spinal cords of rats receivingvehicle alone. FIG. 3A shows cross sections of spinal cords stained witha GFAP antibody to identify activated astrocytes. Panels (a) and (b) arefrom a rat treated with vehicle alone at 5× and 20× magnification,respectively, while panels (c) and (d) are from a rat treated with 100mg/kg DMF, shown at 5× and 20× magnification, respectively.

FIG. 3B shows morphometric quantitation using Apeiro colordeconvolution.

FIG. 3C shows a rat spinal cord cross section with the ventral greymatter and white matter zones indicated. Those zones were selected formorphometry. FIG. 3D shows morphometric quantitation of positive GFAPstaining in ventral grey and white matter.

EXAMPLE 3

This experiment shows that BG00012 reduces activation of primarycultured astrocytes. FIG. 4A shows quantitative PCR analysis of GFAPexpression following DMF stimulation at the indicated concentrations for6 hrs and 24 hrs. As shown, DMF inhibits GFAP expression in aconcentration dependent manner.

FIG. 4B shows quantitative PCR analysis of TNF expression following DMFstimulation at the indicated concentration for 24 hours, with LPS addedat 0, 10, and 30 ng/ml 4 hrs. prior to harvest. As shown, LPS inducesTNF expression in a dose dependent manner and that induced TNFexpression is repressed by DMF in a dose dependent manner.

FIG. 4C shows quantitative PCR analysis of NQO1 expression following DMFstimulation at the indicated concentrations for 6 hrs and 24 hrs. Asshown, DMF induces NQO1 expression in a concentration dependent manner.It is also apparent from the data that NQO1 is induced at a higher levelby exposure to DMF for 24 hrs compared to induction by a 6 hourexposure.

Finally, FIG. 4D shows the results of an MTT assay after 24 hrs ofBG00012 (DMF) or vehicle (DMSO) treatment of astrocytes. The dataindicate that Astrocyte viability is not compromised by BG00012treatment.

EXAMPLE 4

FIG. 5 shows that BG00012 inhibits inflammatory cytokines andpro-inflammatory signaling induced by LPS stimulation of primaryastrocytes. Primary astrocytes were treated with LPS at 0, 10, and 100ng/ml, as indicated, and also treated with DMF at 0, 3, 10, or 30 μM asindicated. The magnitude of expression of each gene under each conditionwas scaled from 0 to 1, so that differences in color represent changesin expression of each gene as the conditions varied. The darkest greencolor represents no detectable expression (0), while the brightest redcolor represents the highest expression measured for that gene (1).Generally speaking, the data show that expression of these genes isincreased by LPS treatment, with all markers showing induction by 100ng/ml LPS treatment. The induced expression of many of the markers wassuppressed by DMF treatment. In particular, strongly suppressedinduction of Ccl20, Ccl3, Ccl4, Cxcl1, Cxcl10, Cxcl2, Cxcl3, Cxcl6,IL1a, II1b, Tnf, Ifit3, and Zc3h12a.

EXAMPLE 5

This example provides data indicating that DMF (BG00012) can inhibitastrogliosis and microglial activation associated with chronic relapsingEAE (crEAE) in rats.

crEAE was induced by intradermal MOG/IFA immunization in Brown Norwayrats. BG00012 was administered orally at a daily interval beginningthree days after immunization. BG00012 reduced average clinical scoresof disease in all treated groups. For the 100 mg/kg treatment group,average disease score at day 28 was 0.71 (n=6, SD=1.17) compared to 2.29(n=6, SD=1.29) for the vehicle group. Immunohistochemistry of lumbarspinal cord sections showed decreased staining of GFAP, a marker foractivated astrocytes, and IBA-1, a marker for activated microglia.

Quantitative PCR of mRNA from spinal cords revealed 52% and 54%decreases in IBA-1 and GFAP mRNAs, respectively, in BG00012 treatedgroup compared to the vehicle treated group.

Direct effects of BG00012 on specific astrocytic and microglial cellswere tested in vitro using primary rat astrocytes and RAW264.7macrophage cells. LPS stimulation in the presence of BG00012 resulted in77% and 59% reduction in TNF-a mRNA in astrocytes and macrophages,respectively. Global gene expression profiling of LPS stimulated cellsshowed that BG00012 can inhibit many pro-inflammatory gene products inboth cell types.

These findings indicate that suppression of reactive gliosis andinhibition of macrophage function may contribute to the therapeuticeffect of BG00012 as a part of its dual anti-inflammatory and CNSneuroprotective mechanism of action.

The data reported herein indicate that BG00012 inhibits disease inrelapsing rat EAE. Histological analysis has shown decreased levels ofastrocyte activation markers in BG00012-treated spinal cords. In vitrodata suggest that BG00012 can directly inhibit activation of astrocytes.Finally, pro-Inflammatory gene expression is reduced following BG00012treatment in LPS stimulated primary astrocytes. These findings point toa role for BG00012 in suppression of reactive gliosis and dualanti-inflammatory and CNS neuroprotective mechanisms of action.

EXAMPLE 6

This Example analyzes the effect of MMF on cultured astrocytes.Specifically, the results show that MMF treatment upregulates cellularreduction potential, reduces H₂O₂-induced Ca⁺⁺ mobilization, and reducesH₂O₂-induced cellular death.

Primary cultures of human spinal cord astrocytes were treated for 24 hrwith a titration of MMF or DMSO as a diluent control. Following 24 hrincubation with compound, cells were washed 1× with growth media andincubated with the cell-permeant substrate monochlorobimane. When boundto glutathione this substrate increases fluorescence. A clearconcentration-dependent increase in cellular glutathione levels wereobserved upon treatment with MMF. FIG. 6A. Similar MMF treated cellswere also incubated with the cell permeant substrate resazurin, whichincreases fluorescence upon reduction to resorufin by cellular redoxmechanisms and is used as a measure of cellular metabolic activity(CellTiter-Blue assay). This assay demonstrates a similar result as in(FIG. 6A), in that there is a clear MMF concentration-dependent increasein the ability of treated cells to reduce the substrate. FIG. 6B. Toensure the increases in glutathione and metabolic activity were notsimply due to cellular proliferation in response to MMF treatment,parallel dishes of cells were incubated with calcein AM. Cellularesterases cleave this molecule to generate a fluorescent metabolite,which provides a measure of cell viability and relative total cellnumbers. No substantial concentration-dependent changes were observed.FIG. 6C. Taken together these data suggest MMF treatment upregulates anantioxidant response in cultured human spinal cord astrocytes, which mayoffer neuroprotective benefit upon oxidative challenge.

In another experiment, primary cultures of human spinal cord astrocyteswere treated for 24 hr with a titration of MMF or DMSO as a diluentcontrol. Following 24 hr incubation with compound, cells were washed 1×with HBSS and incubated with Calcium4 (Molecular Devices) loading dye.This dye permeates into cells and increases fluorescence upon binding offree intracellular Ca⁺⁺. Cells were then challenged with 50 mMH₂O₂ andmonitored for changes in fluorescence . FIG. 7A shows raw fluorescencetraces from cells indicated MMF reduced mobilization of intracellularCa⁺⁺ in a dose-dependent fashion. FIG. 7B shows quantitation of thechange in fluorescence intensity over basline (DRFU) demonstrated that30 mM MMF reduced accumulation of Ca⁺⁺ to background levels (compared tono H₂O₂ control), and protection against the H202 challenge is dosedependent. Fitting this data with non-linear regression reveals EC₅₀=5.4mM. FIG. 7C. These data suggest MMF is able to suppress release ofintracelluar Ca⁺⁺, which may offer neuroprotective benefit by preventinginitiation of downstream apoptotic cascades.

In another experiment, primary cultures of human spinal cord astrocyteswere treated for 24 hr with a titration of MMF or DMSO as a diluentcontrol. Following 24 hr incubation with compound, cells were washed 1×with HBSS and challenged with 500 mM H₂O₂ for two hours, then washed innormal growth media, then incubated for an additional 24 hours. FIG. 8Ashows results of using the same calcein AM technique as in FIGS. 6A-6Cto monitor cell viability, a significant decrease was observed withtransient 500 mM H₂O₂ treatment followed by a 24 hour recovery period.This loss of viability is attenuated by MMF in a concentration-dependentmanner. FIG. 8B shows that similar effects were observed on cellularmetabolism, as measured by cell-dependent reduction of the substrateresazurin. FIG. 8C shows somewhat more modest MMF-dependent protectiveeffects were observed by examining cellular ATP levels, although a clearconcentration-dependent response was observed.

Interestingly, the highest concentration of MMF appeared to reducecellular ATP levels. Taken together, these data suggest that MMFtriggers a response in human spinal cord astrocytes that isneuroprotective against oxidative stress.

EXAMPLE 7

Total cell lysates were prepared from the astrocyte cultures treated asindicated above. Nrf2 was detected by Western blotting. GAPDH wasincluded as a housekeeping protein control. The results shown in FIG. 9show that DMF and MMF increase levels of Nrf2 in primary rat and humanastrocytes.

EXAMPLE 8

Global analysis of gene expression in astrocytes treated with DMF wasperformed using the Affymetrix GeneChip technology. Genes affected byDMF were identified as transcripts whose levels were significantly(p<10⁷) increased in DMF-treated cells compared to the untreatedastrocytes. The resulting gene list was annotated using the IngenuityIPA database. As shown in FIG. 10, DMF activates expression of Nrf2target genes, including genes known to regulate glutathione metabolism.As shown in FIG. 11, modulation of global gene expression by DMFindicates effects on astrocyte functions related to nervous systemdevelopment, function, and disease. Specific genes involved incytoprotection and glutathione metabolism, identified in this assay,include, but are not limited to, Gsta2, Gsta3, Gcic, Ggt1, Txnrd1,Srxn1, Sqstm1, and Nqo1.

EXAMPLE 9

As shown in FIG. 12, DMF treatment diminishes myelin loss during EAE inrat spinal cords. Morphometry analysis of EAE spinal cords was performedwith Aperio software. Histological alterations were quantified withAperio Image Scope software v10.1. The Color Deconvolution algorithm wasused to select intensity thresholds either for brown DAB staining (GFAPand IBA-1 Immunostains) or for bright turquoise blue staining (LuxolFast Blue). Positive staining was determined by the percent of strongpositive stained pixels for each intensity threshold. Three lumbarspinal cord sections were quantitated per rat and the values for percentstrong positive pixels were averaged for the final intensity value.

1-40. (canceled)
 41. A method of treating a subject having Friedreichataxia, the method comprising administering to the subject atherapeutically effective amount of at least one compound of Formula I:

wherein R¹ and R² are independently selected from OH, O⁻, and(C₁₋₆)alkoxy, provided that at least one of R¹ and R² is (C₁₋₆)alkoxy,or a pharmaceutically acceptable salt thereof.
 42. The method of claim41, wherein the at least one compound is chosen from dimethyl fumarateand monomethyl fumarate.
 43. The method of claim 41, wherein the onlyactive agent administered to the subject is dimethyl fumarate.
 44. Themethod of claim 41, wherein the only active agent administered to thesubject is monomethyl fumarate.
 45. The method of claim 41, wherein theonly active agents administered to the subject are dimethyl fumarate andmonomethyl fumarate.
 46. The method of claim 41, wherein the at leastone compound is administered orally.
 47. The method of claim 42, whereinthe at least one compound is administered orally.
 48. The method ofclaim 43, wherein the at least one compound is administered orally. 49.The method of claim 44, wherein the at least one compound isadministered orally.
 50. The method of claim 45, wherein the at leastone compound is administered orally.
 51. The method of claim 46, whereinthe at least one compound is administered in the form of a pill, atablet, a microtablet, a pellet, a micropellet, a capsule, a capsulecontaining microtablets, or a liquid formulation.
 52. The method ofclaim 48, wherein dimethyl fumarate is administered in the form of acapsule containing enteric coated microtablets.